Cryo-EM analyses of dimerized spliceosomes provide new insights into the functions of B complex proteins

The B complex is a key intermediate stage of spliceosome assembly. To improve the structural resolution of monomeric, human spliceosomal B (hB) complexes and thereby generate a more comprehensive hB molecular model, we determined the cryo-EM structure of B complex dimers formed in the presence of ATP\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\gamma$$\end{document}γS. The enhanced resolution of these complexes allows a finer molecular dissection of how the 5′ splice site (5′ss) is recognized in hB, and new insights into molecular interactions of FBP21, SNU23 and PRP38 with the U6/5′ss helix and with each other. It also reveals that SMU1 and RED are present as a heterotetrameric complex and are located at the interface of the B dimer protomers. We further show that MFAP1 and UBL5 form a 5′ exon binding channel in hB, and elucidate the molecular contacts stabilizing the 5′ exon at this stage. Our studies thus yield more accurate models of protein and RNA components of hB complexes. They further allow the localization of additional proteins and protein domains (such as SF3B6, BUD31 and TCERG1) whose position was not previously known, thereby uncovering new functions for B-specific and other hB proteins during pre-mRNA splicing.

showing the rearrangements in the U4/U6 RNA-RNA network and the transfer of the 5'ss from U1 to U6 during B complex formation.In the pre-B complex, the 5' end of the U1 snRNA base pairs with nucleotides at or adjacent to the 5'ss GU dinucleotide.During B complex formation, the DEAD-box helicase PRP28 disrupts the U1/5'ss interaction and facilitates the handover of the 5'ss to the U6 snRNA, leading to formation of an extended U6/5'ss helix.At the same time, nucleotides in loop 1 of the U5 snRNA base pair with pre-mRNA nucleotides directly upstream of the 5'ss GU at the 3' end of the 5' exon.See Fig. EV1H for a more comprehensive depiction of the RNA-RNA network in the human B complex.In the human pre-B complex, the U4 and U6 snRNAs, not only are extensively based-paired via helix I and II, but also form an additional helix (U4/U6 helix III) and the U4 snRNA forms a socalled quasi-pseudoknot.During B complex formation the latter and U4/U6 helix III are disrupted, and U6 nucleotides involved in helix III formation now form part of the extended U6/5'ss helix.C. Cartoon showing the large-scale translocation of the BRR2 helicase domain, comprised of an N-terminal helicase cassette (NC) and C-terminal helicase cassette (CC), during B complex formation.The BRR2 helicase domain is translocated from its position close to the PRP8 reverse transcriptase-like domain in pre-B to the PRP8 endonuclease-like domain in the B complex.In pre-B, the U4 snRNA binding site in the RecA1 and RecA2 domains of the BRR2 N-terminal cassette is blocked by the C-terminal tail of PRP8.In the B complex, the U4 snRNA has bound the RecA domains, leading to an active BRR2 conformation, but additional mechanisms involving other B complex proteins prevent BRR2 from unwinding the U4/U6 helices.The N-terminal HAT repeats of PRP6 are also repositioned during the transformation of pre-B to B, which potentially is triggered by PRP6 phosphorylation by PRP4 kinase (PRP4K).D. The B-specific proteins are recruited, together with TCERG1 and BUD13, during Bcomplex formation.See Appendix Table S1, for a description of the roles of the B-specific proteins in the B complex.The structural rearrangements and compositional changes described in the legend are based on this study and previous cryo-EM studies elucidating the structure of the human pre-B and B complexes (Bertram et al, 2017; Charenton et al, 2019; Zhan et al, 2018).S1.Roles of the B-specific and other selected proteins in the human B complex.The roles of the listed proteins are based primarily on this study and previous cryo-EM studies of the human B complex (Bertram et al, 2017; Zhan et al, 2018).

SMU1
Bridges BRR2 and SF3B3, and helps to stabilize BRR2's new position after its translocation.It also bridges the two B complex protomers at their interface in the B dimer.

RED
Based on crosslinks, RED latches onto U2 proteins and PRP8, including the PRP8 N-terminal domain (NTD), likely stabilizing the U2 snRNP interaction with the tri-snRNP.

PRP38
Acts as a binding hub for the B-specific proteins MFAP1 and SNU23, and tethers them to the PRP8 NTD.PRP38 and associated proteins help to stabilize the half-closed conformation of PRP8.

SNU23
Stabilizes the extended part of the U6/5'ss helix by direct contacts or indirectly via interacting with the PRP8 RH-Jab1 linker.SNU23 interacts with the BRR2 N-terminal helicase cassette (BRR2 NC ) and tethers it to the U6/5'ss helix.

FBP21
Binds to the U6/5'ss helix via its zinc finger and to the RecA2 domain of BRR2 NC via its N-terminal α-helix, and thereby likely inhibits BRR2's movement towards U4/U6 helix I.It also binds via its Cterminal region to BRR2's C-terminal helicase cassette, thereby inhibiting BRR2's helicase activity.
MFAP1 & UBL5 MFAP1 bridges via a long α-helix 291-313 to PRP38, and SNU23, via αhelix  to BRR2 NC and via α-helix  to the WD40 domain of SMU1B, thereby likely stabilizing BRR2 in its new position after translocation. Based o crosslinks, the N-terminal region latches on U2 SF3B1, thus MFAP1 connects SMU1, BRR2 and the 5' domain of U2 with the RNP core of the B complex.A large part of MFAP1's Cterminal region (aa 215-393) forms a globular domain that binds UBL5, and together with it, forms a 5' exon-binding channel.Both proteins also stabilize the 5' exon/U5 loop 1 base pairing interaction.

TCERG1
The six C-terminal FF domains of TCERG1 act as a brace contacting the PRP8 reverse transcriptase-like (RT) domain, SNU114 and, in conjunction with BUD31, the 5' end of U6 snRNA, likely stabilizing this part of the B complex during the BRR2-mediated remodeling of the spliceosome.

PRP8
Acts as a large scaffold that binds numerous B complex proteins and RNA regions.The PRP8 NTD clamps the major stems of the U5 snRNA and stably interacts with SNU114.It also provides docking sites for the extended U6/5'ss helix, and for the PRP38/SNU23/MFAP1 protein complex, as well as BUD13 and the TCERG1 FF1 domain.The PRP8 En domain interacts with the PRP8 Jab1 domain, which in turn stably docks to the N-terminal helicase cassette of BRR2.The PRP8 RH domain interacts with BRR2, SNU66 and the N-terminal HAT domain of PRP6.Amino acid side chains of two loops of the PRP8 Linker region recognize the 5'ss nucleotides G+1 and G-1.The PRP8 helical bundle binds DIM1.Several regions of the PRP8 Large domain interact with U4/U6 RNP proteins.

BRR2
A DEXH-box RNA helicase that unwinds the U4/U6 helix during B act formation.The RecA domains of the N-terminal helicase cassette bind a single stranded region of the U4 snRNA directly upstream of the U4/U6 helix 1.

PRP4
The PRP4 WD40 domain bridges the U4/U6 proteins SNU13 and PRP6, as well as the C-terminal ferredoxin-like domain of PRP3.The N-terminal helical bundle of PRP4 binds PPIH, BRR2, and the central region of SF3A1.

SNU66
Several N-terminally-located α-helices of SNU66 connect the Nterminal HAT domain of PRP6 with the PRP8 RT, RH and NTD domains, and likely stabilize these domains in the B complex after their rearrangements during the pre-B to B transition.
PPIH PPIH binds to the N-terminal region of PRP4 and provides a major docking site for the long N-terminal α-helix of FBP21.It also contacts PRP3.

SF3B1
The SF3B1 HEAT domain clamps the extended U2/BS helix and acts as a binding platform for several other SF3b complex proteins.

SF3B6
The SF3B6 RRM is bound at the C-terminal region of the SF3B1 HEAT domain and is located near U2/U6 helix II.

SF3A1
SF3A1 connects the U2 snRNP with the tri-snRNP core via its central tri-snRNP-interacting region comprised of amino acids 409-489.This region contains three α-helices that dock to PRP4's N-terminal helical bundle and PPIH, bind to the 5' stem-loop of U4 snRNA, and interact via its most C-terminally located α-helix with the U5 protein DIM1.

SF3A2
The SF3A2 b-sandwich domain and the SF3B4 RRM1 domain chaperon the intron region directly upstream of nucleotides forming the branch site helix.Based on crosslinks, the b-sandwich domain may also function as a docking site for the RRM1 of the hnRNP A1 protein.
Appendix Table S2.Protein composition of hB complexes.Proteins were identified by nano UHPLC-ESI MS with at least 2 unique peptides (peptide FDR 0.05%).PSM stands for peptide-spectrum match.The 90 most abundant proteins are shown as judged from a ratio of PSMs to protein molecular weight.PSMs are the sum of three technical replicates.Asterisk marks the recombinant protein used for affinity purification of hB.
Appendix Figure S1.Structural changes during the spliceosomal pre-B to B complex transition.A. Cartoon of the conformational change in PRP8 during the transformation of the pre-B complex into B. For simplicity only selected PRP8 domains are shown.Movement of the PRP8 endonuclease-like (En) domain (indicated by an arrow) toward the PRP8 N-terminal domain (NTD) converts the PRP8 open conformation to a half-closed one in the B complex.During the pre-B to B transition, the PRP8 RNase H (RH) domain also rotates ca 180 degrees.RT, reverse transcriptase-like domain.B. Schematic